WO2023065384A1 - Culture medium for human mesenchymal stem cell - Google Patents
Culture medium for human mesenchymal stem cell Download PDFInfo
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- WO2023065384A1 WO2023065384A1 PCT/CN2021/127005 CN2021127005W WO2023065384A1 WO 2023065384 A1 WO2023065384 A1 WO 2023065384A1 CN 2021127005 W CN2021127005 W CN 2021127005W WO 2023065384 A1 WO2023065384 A1 WO 2023065384A1
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- mesenchymal stem
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- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 46
- 239000001963 growth medium Substances 0.000 title claims abstract description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 10
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- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 8
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- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 claims abstract description 4
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- 102100024785 Fibroblast growth factor 2 Human genes 0.000 claims abstract 2
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 claims description 3
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- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 3
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- 210000004381 amniotic fluid Anatomy 0.000 description 1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
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- C12N2509/10—Mechanical dissociation
Definitions
- the invention relates to a human mesenchymal stem cell culture medium, which belongs to the field of biological materials.
- MSC Mesenchymal stem cells
- mesenchymal stem cells are a kind of pluripotent stem cells that have certain differentiation potential and can differentiate into various cell types such as skeletal muscle, cardiac muscle, fat, cartilage, and bone. It can be used clinically for the replacement of target cell groups and the repair of damaged and diseased tissues and organs. It has the function of immune regulation and can play the function of immune reconstruction. Allograft rejection is mild, and the requirements for matching are not strict, so it is widely used.
- Mesenchymal stem cells mainly exist in bone marrow, and can also be isolated and prepared from fat, synovium, bone, muscle, lung, liver, pancreas and other tissues, as well as amniotic fluid and umbilical cord blood.
- the purpose of the present invention is to overcome the deficiencies of the prior art and provide a culture medium for human mesenchymal stem cells.
- a culture medium for human mesenchymal stem cells including fetal bovine serum: 25ml, insulin: 5mg, transferrin: 2.75mg, fibroblast growth factor-2: 5ug, insulin-like growth factor 2: 5ug, taurine: 0.625mg , Putrescine hydrochloride: 0.5mg, Methyl bD-xylopyranoside: 50mg, ⁇ -ketoglutaric acid: 7.3mg, Bovine serum albumin: 0.625g, Linoleic acid: 2.675mg, Cholesterol: 2.5mg, ⁇ -Tocopherol: final concentration 9 ⁇ 10 -7 M, N-acetylcysteine: 5mg, ascorbic acid: 25mg, sodium selenite: 3.35ug, glycine: 3.75mg, L-alanine: 4.45mg, L-Asparagine: 6.6mg, L-Aspartic Acid: 6.65mg, L-
- the cell culture medium of the present invention can culture human mesenchymal stem cells for more than 30 generations, and the stem cells can still maintain a relatively good shape at the 30th generation, have the ability to proliferate, and can continue to maintain the differentiation properties of human mesenchymal stem cells, that is Possesses the potential to differentiate into fat, cartilage and bone.
- Figure 1 is the morphology of cultured human mesenchymal stem cells
- Figure 2 is the proliferation curve of human mesenchymal stem cells
- Figure 3 is the differentiation ability of human mesenchymal stem cells.
- DMEM/F12 Dulbecco's modified Eagle medium/nutrient mixture F-12 (manufacturer: Thermo Fisher); other raw materials are commercially available.
- Embodiment 1 A culture medium for human mesenchymal stem cells, including fetal bovine serum: 25ml, insulin: 5mg, transferrin: 2.75mg, fibroblast growth factor-2: 5ug, insulin-like growth factor 2: 5ug, taurine Acid: 0.625mg, putrescine hydrochloride: 0.5mg, methyl bD-xylopyranoside: 50mg, ⁇ -ketoglutaric acid: 7.3mg, bovine serum albumin: 0.625g, linoleic acid: 2.675mg, cholesterol: 2.5mg, ⁇ -tocopherol: final concentration of 9 ⁇ 10 -7 M, N-acetylcysteine: 5mg, ascorbic acid: 25mg, sodium selenite: 3.35ug, glycine: 3.75mg, L-alanine : 4.45mg, L-Asparagine: 6.6mg, L-Aspartic Acid: 6.65m
- the umbilical cord was washed thoroughly with saline and cut into small pieces. Cut the umbilical cord longitudinally, and use forceps to peel off two umbilical arteries and one umbilical vein in the umbilical cord. The umbilical cord was cut into small pieces, uniformly inoculated in a T75 culture flask containing 15 mL of a human mesenchymal stem cell culture medium in Example 1, and cultured at 37° C. with 5% carbon dioxide, and the cells obtained were P0 generation cells.
- the replacement method is to suck up the old medium with a Pasteur pipette and pipette Slowly add an equal amount of new human mesenchymal stem cell culture medium preheated at 37°C to the tube.
- the specific operation method is:
- Figure 1 is the morphology of cultured human mesenchymal stem cells, in which:
- Figure 2 is the proliferation curve of human mesenchymal stem cells.
- the rhombus point represents the proliferation curve of cells cultured in human mesenchymal stem cell medium (series 1)
- the circle point represents DMEM+10% FBS (series 2).
- the human mesenchymal stem cells cultured in the human mesenchymal stem cell medium still proliferate well at the 30th passage, and the human mesenchymal stem cells cultured in DMEM+10% FBS have lost their proliferative ability at the 7th passage.
- Figure 3 is the differentiation ability of human mesenchymal stem cells.
- Shown in the figure is a picture of induced differentiation of human mesenchymal stem cells cultured in human mesenchymal stem cell culture medium to the 30th passage or later.
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Abstract
A culture medium for a human mesenchymal stem cell, comprising fetal bovine serum, insulin, transferrin, fibroblast growth factor-2, insulin-like growth factor 2, taurine, putrescine hydrochloride, methyl b-D-xylopyranoside, α-ketoglutaric acid, bovine serum albumin, linoleic acid, cholesterol, α-tocopherol, N-acetylcysteine, ascorbic acid, sodium selenite, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-serine, L-proline, sodium pyruvate and DMEM/F12. The human mesenchymal stem cells cultured with the cell culture medium can subculture for more than 30 generations, and can still keep a relatively good form and has a proliferation ability in the 30th generation, and the differentiation property of the human mesenchymal stem cell can be continuously maintained, that is, the human mesenchymal stem cell has the potential to differentiate into fat, cartilage and bone cell.
Description
本发明涉及一种人间充质干细胞培养基，属于生物材料领域。The invention relates to a human mesenchymal stem cell culture medium, which belongs to the field of biological materials.
间充质干细胞(MSC,mesenchymal stem cells)是一种多能干细胞，具有一定的分化潜能，可以分化为骨骼肌、心肌、脂肪、软骨、骨骼等多种细胞类型。应用于临床，可用于目标细胞群的替换和损伤病变组织器官的修复。具有免疫调节功能，可以发挥免疫重建的功能。异体移植排异反应较轻，对配型的要求不严格，因而应用广泛。Mesenchymal stem cells (MSC, mesenchymal stem cells) are a kind of pluripotent stem cells that have certain differentiation potential and can differentiate into various cell types such as skeletal muscle, cardiac muscle, fat, cartilage, and bone. It can be used clinically for the replacement of target cell groups and the repair of damaged and diseased tissues and organs. It has the function of immune regulation and can play the function of immune reconstruction. Allograft rejection is mild, and the requirements for matching are not strict, so it is widely used.
间充质干细胞主要存在于骨髓中，也能够从脂肪、滑膜、骨骼、肌肉、肺、肝、胰腺等组织以及羊水、脐带血中分离和制备间充质干细胞。Mesenchymal stem cells mainly exist in bone marrow, and can also be isolated and prepared from fat, synovium, bone, muscle, lung, liver, pancreas and other tissues, as well as amniotic fluid and umbilical cord blood.
分离人间充质干细胞后，需要培养人间充质干细胞到足够量，用于分化为目标组织。也需要在培养过程中保持分化潜能。我们发现用传统细胞培养基，以(DMEM+10％FBS)为例，培养人间充质干细胞，细胞到第三代就会出现明显的衰老特征，因此，亟需一种能较多代数培养人间充质干细胞，并且保持其分化潜能的培养基。After isolating human mesenchymal stem cells, it is necessary to culture human mesenchymal stem cells to a sufficient amount for differentiation into target tissues. There is also a need to maintain differentiation potential during culture. We found that using traditional cell culture medium (DMEM+10%FBS) as an example to cultivate human mesenchymal stem cells, the cells will show obvious aging characteristics at the third generation. Mesenchymal stem cells and a medium for maintaining their differentiation potential.
发明内容Contents of the invention
本发明的目的是克服现有技术的不足，提供一种人间充质干细胞培养基。The purpose of the present invention is to overcome the deficiencies of the prior art and provide a culture medium for human mesenchymal stem cells.
本发明的技术方案概述如下：Technical scheme of the present invention is summarized as follows:
一种人间充质干细胞培养基，包括胎牛血清：25ml、胰岛素：5mg、转铁蛋白：2.75mg、成纤维生长因子-2：5ug、胰岛素样生长因子2：5ug、牛磺酸：0.625mg、盐酸腐胺：0.5mg、甲基b-D-木吡喃糖苷：50mg、α-酮戊二酸：7.3mg、牛血清白蛋白：0.625g、亚油酸：2.675mg、胆固醇：2.5mg、α-生育酚：终浓度为9×10
-7M、N-乙酰半胱氨酸：5mg、抗坏血酸：25mg、亚硒酸钠：3.35ug、甘氨酸：3.75mg、L-丙氨酸：4.45mg、L-天门冬酰胺：6.6mg、L-天门冬氨酸：6.65mg、L-谷氨酸：7.35mg、L-丝氨酸：5.25mg、L-脯氨酸：5.75mg、丙酮酸钠：27.5mg，用DMEM/F12补齐到500ml。
A culture medium for human mesenchymal stem cells, including fetal bovine serum: 25ml, insulin: 5mg, transferrin: 2.75mg, fibroblast growth factor-2: 5ug, insulin-like growth factor 2: 5ug, taurine: 0.625mg , Putrescine hydrochloride: 0.5mg, Methyl bD-xylopyranoside: 50mg, α-ketoglutaric acid: 7.3mg, Bovine serum albumin: 0.625g, Linoleic acid: 2.675mg, Cholesterol: 2.5mg, α -Tocopherol: final concentration 9×10 -7 M, N-acetylcysteine: 5mg, ascorbic acid: 25mg, sodium selenite: 3.35ug, glycine: 3.75mg, L-alanine: 4.45mg, L-Asparagine: 6.6mg, L-Aspartic Acid: 6.65mg, L-Glutamic Acid: 7.35mg, L-Serine: 5.25mg, L-Proline: 5.75mg, Sodium Pyruvate: 27.5mg , make up to 500ml with DMEM/F12.
本发明的优点：Advantages of the present invention:
本发明细胞培养基培养人间充质干细胞，可以传代培养30代以上，到第30代所述干细胞仍然能保持比较好的形态，具有增殖能力，并能继续维持人间充质干细胞的分化属性，即具备向脂肪、软骨和骨的分化潜能。The cell culture medium of the present invention can culture human mesenchymal stem cells for more than 30 generations, and the stem cells can still maintain a relatively good shape at the 30th generation, have the ability to proliferate, and can continue to maintain the differentiation properties of human mesenchymal stem cells, that is Possesses the potential to differentiate into fat, cartilage and bone.
图1为培养的人间充质干细胞形态；Figure 1 is the morphology of cultured human mesenchymal stem cells;
图2为人间充质干细胞增殖曲线；Figure 2 is the proliferation curve of human mesenchymal stem cells;
图3为人间充质干细胞分化能力。Figure 3 is the differentiation ability of human mesenchymal stem cells.
DMEM/F12：杜尔贝科改良Eagle培养基/营养混合物F-12(生产厂家：赛默飞)；其 它原料均为市售。DMEM/F12: Dulbecco's modified Eagle medium/nutrient mixture F-12 (manufacturer: Thermo Fisher); other raw materials are commercially available.
下面通过具体实施例对本发明作进一步的说明。The present invention will be further described below by specific examples.
实施例1、一种人间充质干细胞培养基，包括胎牛血清：25ml、胰岛素：5mg、转铁蛋白：2.75mg、成纤维生长因子-2：5ug、胰岛素样生长因子2：5ug、牛磺酸：0.625mg、盐酸腐胺：0.5mg、甲基b-D-木吡喃糖苷：50mg、α-酮戊二酸：7.3mg、牛血清白蛋白：0.625g、亚油酸：2.675mg、胆固醇：2.5mg、α-生育酚：终浓度为9×10
-7M、N-乙酰半胱氨酸：5mg、抗坏血酸：25mg、亚硒酸钠：3.35ug、甘氨酸：3.75mg、L-丙氨酸：4.45mg、L-天门冬酰胺：6.6mg、L-天门冬氨酸：6.65mg、L-谷氨酸：7.35mg、L-丝氨酸：5.25mg、L-脯氨酸：5.75mg、丙酮酸钠：27.5mg，用DMEM/F12补齐到500ml。
Embodiment 1. A culture medium for human mesenchymal stem cells, including fetal bovine serum: 25ml, insulin: 5mg, transferrin: 2.75mg, fibroblast growth factor-2: 5ug, insulin-like growth factor 2: 5ug, taurine Acid: 0.625mg, putrescine hydrochloride: 0.5mg, methyl bD-xylopyranoside: 50mg, α-ketoglutaric acid: 7.3mg, bovine serum albumin: 0.625g, linoleic acid: 2.675mg, cholesterol: 2.5mg, α-tocopherol: final concentration of 9×10 -7 M, N-acetylcysteine: 5mg, ascorbic acid: 25mg, sodium selenite: 3.35ug, glycine: 3.75mg, L-alanine : 4.45mg, L-Asparagine: 6.6mg, L-Aspartic Acid: 6.65mg, L-Glutamic Acid: 7.35mg, L-Serine: 5.25mg, L-Proline: 5.75mg, Pyruvate Sodium: 27.5mg, make up to 500ml with DMEM/F12.
实施例2Example 2
人间充质干细胞的培养：Culture of human mesenchymal stem cells:
(1)P0代细胞的获得：(1) Acquisition of P0 generation cells:
人脐带组织贴壁法分离人间充质干细胞。用生理盐水充分洗涤脐带，并剪成小段。将脐带纵向剪开,用镊子剥去脐带中的两根脐动脉和一根脐静脉。将脐带剪碎，均匀接种于装有15mL实施例1一种人间充质干细胞培养基的T75培养瓶中，37℃，5％二氧化碳培养，得到的细胞即为P0代细胞。Isolation of human mesenchymal stem cells from human umbilical cord tissue. The umbilical cord was washed thoroughly with saline and cut into small pieces. Cut the umbilical cord longitudinally, and use forceps to peel off two umbilical arteries and one umbilical vein in the umbilical cord. The umbilical cord was cut into small pieces, uniformly inoculated in a T75 culture flask containing 15 mL of a human mesenchymal stem cell culture medium in Example 1, and cultured at 37° C. with 5% carbon dioxide, and the cells obtained were P0 generation cells.
(2)换液(2) Liquid replacement
根据步骤(1)的最终的培养基颜色(由原来的红色变成淡黄色)决定更换新的一种人间充质干细胞培养基，更换方法是用巴氏吸管吸掉旧培养基，用移液管，缓慢加入37℃预热的等量新的一种人间充质干细胞培养基。According to the final color of the medium in step (1) (from the original red to light yellow), it is decided to replace a new human mesenchymal stem cell medium. The replacement method is to suck up the old medium with a Pasteur pipette and pipette Slowly add an equal amount of new human mesenchymal stem cell culture medium preheated at 37°C to the tube.
(3)传代(3) Passage
待细胞密度在85％以上时，分瓶培养即为传代。传代1次即为P1代细胞，以此类推。When the cell density is above 85%, culture in separate flasks is subculture. Passage 1 is the P1 generation cells, and so on.
具体操作方法为：The specific operation method is:
a吸掉旧培养基，用37℃预热的pH＝7.4的PBS洗2遍细胞；a Aspirate off the old medium, and wash the cells twice with PBS with pH=7.4 preheated at 37°C;
b每瓶加TrypLE
TM Express胰酶(生产厂家：赛默飞)3ml，室温消化细胞1分钟左右，待细胞变圆后，每瓶加实施例1制备的人间充质干细胞培养基10ml；
b Add 3ml of TrypLETM Express trypsin (manufacturer: Thermo Fisher) to each bottle, digest the cells at room temperature for about 1 minute, and after the cells become round, add 10ml of the human mesenchymal stem cell culture medium prepared in Example 1 to each bottle;
c吸出细胞悬液至50ml离心管中，1000rpm，离心3min，弃上清；c Aspirate the cell suspension into a 50ml centrifuge tube, centrifuge at 1000rpm for 3min, discard the supernatant;
d用10ml一种人间充质干细胞培养基重悬沉淀，经细胞筛过滤，5ml人间充质干细胞培养基冲洗筛网，计数；d Use 10ml of a human mesenchymal stem cell medium to resuspend the pellet, filter through a cell sieve, wash the sieve with 5ml of human mesenchymal stem cell medium, and count;
e调整细胞浓度，根据实际需要铺板或瓶，置于37℃、5％二氧化碳培养箱中培养。e Adjust the cell concentration, plate or bottle according to actual needs, and culture in a 37°C, 5% carbon dioxide incubator.
见图1、2和3。See Figures 1, 2 and 3.
图1为培养的人间充质干细胞形态，其中：Figure 1 is the morphology of cultured human mesenchymal stem cells, in which:
A.DMEM+10％FBS培养人间充质干细胞第3代细胞形态，第3代开始，可以观察到细胞体积增大，出现明显的细胞衰老特征；A. DMEM+10% FBS cultured human mesenchymal stem cells in the third generation of cell morphology. From the third generation, it can be observed that the cell volume increases and the obvious characteristics of cell aging appear;
B.人间充质干细胞培养基培养人间充质干细胞第1代细胞形态；B. Cell morphology of the first generation of human mesenchymal stem cells cultured in human mesenchymal stem cell medium;
C.人间充质干细胞培养基培养人间充质干细胞第3代细胞形态；C. Cell morphology of the third generation of human mesenchymal stem cells cultured in human mesenchymal stem cell medium;
D.人间充质干细胞培养基培养人间充质干细胞第30代细胞形态；细胞在第30代依然维持良好的梭形形态，没有明显的细胞衰老特征。D. The cell morphology of human mesenchymal stem cells cultured in human mesenchymal stem cell medium at passage 30; the cells still maintain a good spindle shape at passage 30, without obvious characteristics of cell senescence.
图2为人间充质干细胞增殖曲线。Figure 2 is the proliferation curve of human mesenchymal stem cells.
其中，菱形点表示人间充质干细胞培养基(系列1)培养细胞的增殖曲线，圆形点表示DMEM+10％FBS(系列2)。如图2中所示，人间充质干细胞培养基培养的人间充质干细胞到第30代仍然增殖很好，DMEM+10％FBS培养的人间充质干细胞到第7代已经丢失增殖力。Among them, the rhombus point represents the proliferation curve of cells cultured in human mesenchymal stem cell medium (series 1), and the circle point represents DMEM+10% FBS (series 2). As shown in Figure 2, the human mesenchymal stem cells cultured in the human mesenchymal stem cell medium still proliferate well at the 30th passage, and the human mesenchymal stem cells cultured in DMEM+10% FBS have lost their proliferative ability at the 7th passage.
图3为人间充质干细胞分化能力。Figure 3 is the differentiation ability of human mesenchymal stem cells.
图中所示为人间充质干细胞培养基培养的人间充质干细胞到第30代以后的细胞诱导分化的图片。Shown in the figure is a picture of induced differentiation of human mesenchymal stem cells cultured in human mesenchymal stem cell culture medium to the 30th passage or later.
A未进行成脂肪诱导，进行油红O染色(脂肪会被染成红色)；A. Without adipogenic induction, oil red O staining was performed (fat will be stained red);
B成脂肪诱导，进行油红O染色(脂肪会被染成红色)；B into fat induction, oil red O staining (fat will be stained red);
C未进行成骨诱导，进行茜素红染色(骨会被染成红色)；C did not perform osteogenesis induction, and performed Alizarin red staining (the bone will be stained red);
D成骨诱导，进行茜素红染色(骨会被染成红色)；D Osteogenic induction, alizarin red staining (bone will be stained red);
E未进行成软骨诱导，进行阿利新蓝染色(软骨会被染成蓝色)；E did not perform chondrogenic induction, and performed Alcian blue staining (cartilage will be stained blue);
F成软骨诱导，进行阿利新蓝染色(软骨会被染成蓝色)。F into chondrogenic induction, perform Alcian blue staining (cartilage will be stained blue).
Claims (1)
- 一种人间充质干细胞培养基，其特征是包括胎牛血清：25ml、胰岛素：5mg、转铁蛋白：2.75mg、成纤维生长因子-2：5ug、胰岛素样生长因子2：5ug、牛磺酸：0.625mg、盐酸腐胺：0.5mg、甲基b-D-木吡喃糖苷：50mg、α-酮戊二酸：7.3mg、牛血清白蛋白：0.625g、亚油酸：2.675mg、胆固醇：2.5mg、α-生育酚：终浓度为9×10 -7M、N-乙酰半胱氨酸：5mg、抗坏血酸：25mg、亚硒酸钠：3.35ug、甘氨酸：3.75mg、L-丙氨酸：4.45mg、L-天门冬酰胺：6.6mg、L-天门冬氨酸：6.65mg、L-谷氨酸：7.35mg、L-丝氨酸：5.25mg、L-脯氨酸：5.75mg、丙酮酸钠：27.5mg，用DMEM/F12补齐到500ml。 A culture medium for human mesenchymal stem cells, characterized by including fetal bovine serum: 25ml, insulin: 5mg, transferrin: 2.75mg, fibroblast growth factor-2: 5ug, insulin-like growth factor 2: 5ug, taurine : 0.625mg, putrescine hydrochloride: 0.5mg, methyl bD-xylopyranoside: 50mg, α-ketoglutaric acid: 7.3mg, bovine serum albumin: 0.625g, linoleic acid: 2.675mg, cholesterol: 2.5 mg, α-tocopherol: final concentration of 9×10 -7 M, N-acetylcysteine: 5mg, ascorbic acid: 25mg, sodium selenite: 3.35ug, glycine: 3.75mg, L-alanine: 4.45mg, L-Asparagine: 6.6mg, L-Aspartic Acid: 6.65mg, L-Glutamic Acid: 7.35mg, L-Serine: 5.25mg, L-Proline: 5.75mg, Sodium Pyruvate : 27.5mg, filled to 500ml with DMEM/F12.
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| CN114958734B (en) | 2023-03-24 |
| CN114958734A (en) | 2022-08-30 |
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